Vertebrate locomotion depends on the formation of ordered connections between spinal motor neurons (MNs) and their target muscles in the limb. Two steps in MN differentiation are critical to the formation of appropriate patterns of limb connectivity: i. the specification of a lateral motor column (LMC), and ii. the generation of motor pool identities within the LMC. Each pool of MNs is clustered at a stereotyped location within the LMC, and projects axons to a single muscle target. Defining how MNs acquire their columnar and pool identities and how their axons are directed to specific muscle targets may provide more general insights into the logic of neural circuit formation in the vertebrate central nervous system. We have obtained evidence that the activity of Hox class transcription factors is involved in specifying MN columnar identity. But many aspects of the link between Hox function, MN diversification and target connectivity remain unexplored. The aim of this proposal is to clarify the contribution of Hox proteins and their downstream effectors to the generation of MN diversity and target connectivity. The first aim will explore how Hox activities determine distinct forelimb and hindlimb LMC fates, whether Hox proteins have roles in the assignment of intrasegmental columnar fate, and the involvement of a Hox independent pathway of MN columnar specification. The second aim will explore whether Hox proteins have functions in motor pool specification, asking how distinct motor pool identities are imposed at different rostrocaudal levels of the LMC, and whether Hox proteins and their co-factors drive MN pool diversity at a single segmental level of the LMC. The third aim will explore relevant targets of Hox proteins, and their possible role in establishing motor axon trajectories in the limb. In particular, we will use mouse genetic methods to test the contribution of Nkx6, POU and Runx class transcription factors to the formation of specific muscle nerve trajectories. Together, these studies should help to define the link between mechanisms of early neuronal specification and the formation of selective connections in a classical vertebrate motor circuit.